Project Summary Sickle cell disease (SCD) carries significant morbidity as a result of red blood cell (RBC) sickling and hemolysis. Stroke is one of the most devastating sequelae of SCD. Chronic transfusion therapy (CTT) reduces stroke risk by (1) supplying normal, non-sickle RBC to circulation, thereby reducing the percentage of endogenous sickle RBC in circulation, and (2) maintaining a higher hemoglobin (Hb), thereby suppressing erythropoiesis of new sickle RBC. While the efficacy of CTT in stroke prophylaxis is well-established, nearly 45% of children continue to have silent or overt strokes despite CTT. The failure of CTT to prevent stroke events may be related to inadequate reduction of circulating sickle RBC and erythropoiesis. The amount of circulating sickle-RBC is related to the survival kinetics of both transfused RBC and endogenous sickle RBC. In a large, longitudinal analysis of CTT in SCD, we found wide variation in the survival of donor RBC following transfusion, with faster clearance associated with patient immune features (historical RBC alloimmunization and spleen presence) and with donor RBC glucose-6-phosphate-dehydrogenase (G6PD) deficiency. To better understand the roles of patient and donor factors in the survival and clearance of transfused RBC, we propose a mechanistic, clinical trial during chronic transfusion episodes in patients with SCD, in which a small aliquot of each transfused unit is labeled with biotin conjugated to RBC surface proteins, to safely identify and measure the in vivo survival of donor RBC. Aim 1 will examine the relationships of the recipient?s immune system (past alloimmunization, splenic volume, and markers of reticuloendothelial system function) on the post-transfusion survival of biotin-labeled donor RBC. Aim 2 will examine the relationships of donor RBC G6PD levels and donor RBC metabolomics with the in vivo survival and changes in donor RBC senescence markers. Completion of these aims will increase our understanding of mechanisms for the variability in RBC survival during CTT, identifying donor and recipient risk factors for decreased RBC survival. Ultimately this knowledge will inform the management of CTT to improve the prevention of strokes in SCD. The applicant, Dr. Yee, is a pediatric hematologist and an emerging clinical researcher in SCD, with a master?s degree in clinical research and experience with pilot and prospective studies of patient-oriented research in SCD. Dr. Yee has identified an exceptional mentorship team with expertise in RBC survival studies and biotinylation, donor RBC metabolomics, and clinical research in SCD. The candidate's short-term career goals for this K23 application are to 1) Develop formal expertise in transfusion medicine; 2) Expand expertise in conducting prospective, interventional clinical research in pediatric SCD and transfusion medicine; 3) Learn the use and interpretation of biotinylation of RBC transfusion to study transfusion survival in vivo. The candidate's long-term career goals are to improve transfusion and cellular therapies with a personalized approach to blood transfusion in children and adults with SCD and to improve the clinical outcomes of transfusion and other SCD therapies while decreasing toxicity and adverse events.